Immunoglobulins conjugated to a drug of interest, generally known as antibody drug conjugates (ADCs), are a promising area of therapeutic research. Recent developments in ADC technology have focused on linker technology that provides for intracellular cleavage or more recently, non-cleavable linkers that provide greater in vivo stability and reduced toxicity. The feasibility of a non-cleavable linker-based approach, however, may be more dependent on the cellular target than in the case of cleavable linkers. ADCs with noncleavable linkers must be internalized and degraded within the cell, whereas compounds with cleavable linkers may be active against targets that are poorly internalized through extracellular drug release and drug entry into tumor cells. Similarly, killing of bystander antigen-negative cells through targeting of antigen-positive cells (collateral toxicity) is presumably only possible with cleavable linkers. As a consequence, it is generally believed that no general linker design exists for all ADCs and that each antibody must be examined separately. Additionally, the efficacy of a drug linked to a toxin may vary, e.g. depending on the cell type or particular tumor cell, such that it may also be necessary to test a variety of drugs against a given target and further in combination with a particular linker system. Development of ADCs therefore remains an expensive and time-consuming process and there is a need in the field for improved linker systems.
Transglutaminases (TGases) have been exploited for some time in the food industry for their ability to cross-link proteins. Such utilization has avoided the need to cross-link in quantitative or stoichiometric fashion. TGases have been shown to be capable of conjugating glutamine and lysine residues, including on antibodies (see, e.g., Josten et al. (2000) J. Immunol. Methods 240, 47-54; Mindt et al (2008) Bioconjug. Chem. 19, 271-278; Jeger et al (2010) Angew. Chem. Int. Ed. 49: 9995-9997); Kamiya et al (2003) Enzyme. Microb. Technol. 33, 492-496, PCT publication WO2012/059882 and US patent publication no. 2011/0184147. While previous attempts to cross-link proteins have studied protein motifs that gave rise to conjugation and identified peptides that can be conjugated, the rules which govern selection by TGases of glutamine residues for modification are still largely unknown. Additionally, little is known about TGases' ability to take up different substrates, or their effect on the ability to TGases to conjugate in quantitative fashion.